This is a request for 5 years of renewed funding to study the molecular basis of novel genetic forms of congenital hyperinsulinism (HI) in children. Previous years focused on the mechanisms of the hyperinsulinism / hyperammonemia syndrome associated with dominant, gain of function mutations of glutamate dehydrogenase (GDH). The goal of this proposal is to determine the mechanisms of HI caused by recessive, loss of function mutations of the mitochondrial fatty acid [unreadable]-oxidation enzyme, short-chain 3-hydroxy acyl-CoA dehydrogenase (SCHAD). Our hypotheses are (1) that the dysregulation of insulin secretion in SCHAD deficiency is caused by an accumulation of a specific fatty acid metabolite(s); and (2) that, unlike other HI disorders which act on the KATP "triggering" pathway of insulin release via plasma membrane depolarization, SCHAD deficiency acts on an "amplification" site(s) downstream of the "triggering" mechanism. These hypotheses will be examined using a mouse SCHAD-/- knockout model. Aim 1 will determine the responses of SCHAD-/- mice to fasting and glucose loading in vivo and whether these responses are altered by a high fat diet. Aim 2 will define the abnormalities in insulin responses to nutrient and glyburide stimulation in the presence and absence of short, medium, and long-chain fatty acids using perifused isolated islets from SCHAD-/- mice and other HI mouse models. Inhibitors of other steps in mitochondrial fatty acid oxidation will be used to test whether effects are specific to SCHAD substrates. Aim 3 will determine the profiles of fatty acid metabolites in isolated SCHAD-/- islets using mass spectrometry to measure acyl-CoAs, acyl-carnitines, and free fatty acids. Aim 4 will determine the effects of SCHAD deficiency on islet cytosolic calcium and mitochondrial energy responses to nutrient stimuli in the presence and absence of fatty acids. Fatty acids are considered to play important roles in regulating insulin secretion by pancreatic [unreadable]-cells, however, their mechanisms of action are poorly understood. SCHAD deficiency provides a unique "experiment of nature" for elucidating not only how fatty acids disturb insulin secretion in children affected with this disorder, but also how these important nutrients contribute to the control of insulin secretion in normal and diabetic humans. [unreadable] [unreadable] [unreadable]